Climate change and the eventual depletion of the world's fossil raw materials reserves are threatening sustainable development [1, 2]. Renewable resources display a large potential for the substitution of chemical compounds derived from petrochemicals. They allow a more sustainable chemistry with the attempt to design chemical products and processes that reduce or eliminate the use and generation of hazardous substances, minimize waste and energy consumption, favor renewable resources and integrate aspects of recycling [3]. Besides, nature offers a wide range of resources mainly from plants due to fast biomass building with low efforts. At the moment only a few industries are using this immense reservoir of resources [4]. Additionally there have been 12 principles postulated for a Green Engineering concerning new processes or the displacement of antiquated processes to engage in a more sustainable development [5]. Several programs were initiated by the European Union and miscellaneous German institutions that promote the research and development concerning this topic.
Succinic acid is a chemical substance with a broad area of application in the chemical industry. Succinic acid represents an important building block that can be converted into various valuable compounds [6]. Beyond fossil based chemistry, derivatives of succinic acid are announced to have a potential of hundreds of thousands tons [7]. Succinic acid is an intermediate of the TCA cycle (tricarboxylic acid cycle) and one of the fermentation end-products of anaerobic metabolism. The research for biotechnological production processes mainly focused on a whole-cell approach using natural overproducers or recombinant producers. The downstream purification cost for fermentation-based processes normally amounts to more than 60% of the total production costs. For succinic acid purification, the separation of byproducts has a crucial effect on process cost [4].
1,4-Butanediol (BDO) is a four carbon dialcohol that is at the moment manufactured exclusively through various petrochemical routes. BDO represents a chemical building block which can be used for production of gamma-butyrolactone (GBL), tetrahydrofuran (THF), pyrrolidone, N-methylpyrrolidone (NMP) and N-vinyl-pyrrolidone [8]. Presently this family has a market opportunity that exceeds 3.000 M. Approximately 1.4 M t BDO is produced by chemical catalyst [8]. The demand for BDO stems largely from its use as an intermediate for polybutylene terephthalate (PBT) plastic resins, polyurethane thermoplastics and co-polyester ethers. BDO also serves as a primary precursor to THF, which is employed as an intermediate for poly(tetramethylene glycol) PTMEG copolymers required for lycra and spandex production. Approximately 0.32 M t of THF is produced globally per year with an annual growth rate over 6%. A significant percentage of growth (>30%) for both BDO and THF is occurring in Asia (China and India). GBL currently is a smaller volume (0.18 M t/year) product which has numerous applications as a solvent, as an additive for inks, paints, and dyes, as well as the primary precursor to pyrrolidone derivatives such as NMP.
However, the replacement of fossil raw materials by biogenic resources is still one of the major obstacles preventing widespread commercialization of such devices.
Enzymes exhibit a great advantage compared to chemical catalysts because they are accepting a wide array of complex molecules as substrates, catalyzing reactions with unparalleled chiral (enantio-) and positional (regio-) selectivities. For this reason, the need of tedious blocking and deblocking steps known in traditional organic synthesis is dispensable [9]. Biological catalysts allow the development of sustainable technologies for the production of chemicals by waste reduction using solvent-free reaction media and minimizing the amount of unrequested by-products complicating the downstream processing [10]. Biocatalyst can be used either as isolated enzymes or in the form of whole cell preparations. The use depends on the requirements of the production process like the half-life of the biocatalyst or the dependency on co-factors. Reaction processes regarding co-factors, especially NAD(P)+, which are utilized in stochimetric quantities whole-cell fermentation is favored. The use of isolated co-factor depending enzymes for establishing a multi-step substrate conversion requires an additional co-factor recycling system for a continuous reaction. Presently, there are some co-factor recycling systems like glucose dehydrogenase/glucose established which allow TTN's (total turnover number) from 103 to 106 or higher for an economical efficient reaction process. It is, however, preferred that cofactor recycling can be achieved without additional substrates.
Carbohydrates represent 95% of the annually renewable biomass. Being renewable carbohydrates such as glucose or other monosaccharides have the potential to compensate the emerging lack of petroleum for the production of bulk chemicals or biofuels. For use as chemicals or fuel carbohydrates contain too many polar functional groups. In the past this was the reason they were disqualified as well-suited precursors for applications in organic chemistry [11]. The use of low molecular weight carbohydrates as well as high molecular weight carbohydrates as the C-source for fermentation processes to produce industrial important chemical compounds is well known. Succinic acid or 2,3-butanediol are two examples of compounds produced by fermentation from carbohydrates. In contrast, the specific conversion of glucose with a multi-step cell free biocatalytic or catalytic process into chemical intermediates is mostly undeveloped. The only economically viable examples are the hydrogenation of glucose to sorbitol followed by the conversion to isosorbide and the oxidation of glucose to gluconate. For the production of C4-compounds from hexoses to date only fermentative processes have been developed, mostly aiming at succinic acid. The Department of Energy of the US has proposed 1,4-diacids, and particularly succinic acid, as key biologically-produced intermediates for the manufacture of the butanediol family of products [6]. However, using fermentation processes, always side products are formed due to the presence of many different enzymes within the organisms. In addition the conditions of the production process (temperature, pH, salt etc.) are limited by the viability of the cells. Product purification often is the most costly process step in a fermentative production system. All these difficulties can be diminished when a cell free production process can be used. By limiting the number of enzymes in such a cell free production to only those essential for the targeted conversion, fewer side products are formed. By applying conditions far from being ambient (e.g. high temperatures, co-solvents) product purification can be more easily integrated into the conversion process. The known pathways from glucose to bifunctional C4-compounds, modified at position 1 and 4, all go via succinate and have never been used in cell free production systems and are probably too difficult to handle (>>10 enzymes) to ever be used in a cell free production system. There is a need for a production process lacking live organisms using just enzymes or other catalysts to cheaply convert hexoses to bifunctional C4 compounds and therefore, there is a need of new and simpler enzymatic pathways, requiring fewer enzymes than existing natural pathways. There is a demand for a new enzymatic pathway that can be applied using purified enzymes or enzymes in cell lysates for a completely cell free in vitro production process or in whole cells containing the enzymes. In addition, it would be beneficial if a new enzymatic pathway could help to improve the yield and productivity of a fermentation process when the enzymes of the pathway are recombinantly expressed in microorganisms.
It is desirable to have such a synthetic pathway for the production of C4 chemicals by alternative means not only to substitute petroleum-based feedstocks but also to facilitate a sustainable process with less waste.
All previously described microbial routes for the production of bifunctional C4-chemicals like 1,4-butanediol or 1,4-aminobutane from C6 polyols and hexoses use more than 10 enzymes in complex metabolic pathways (glycolysis, TCA cycles) requiring a multitude of cofactors (at least NAD+/NADH, ATP/ADP, Coenzyme A) and break down the C6-molecules in two C3-molecules like 3-phospho-glycerate to then reconstruct the C4 entity.